Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Summary

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

Abstract

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.

This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Introduction

Before the last two decades, the treatment of human cancers relied heavily on radiotherapy and chemotherapeutic agents that broadly targeted rapidly proliferating cellular populations by damaging DNA or inhibiting DNA synthesis. Although these approaches did inhibit cancer cell growth, they also had deleterious side effects on normal rapidly proliferating cell types such as intestinal epithelial cells and hair follicle cells. More recently, cancer therapy has begun to utilize chemotherapeutic agents that precisely target proteins within signaling pathways that are critically important for the growth of an individual patient's neoplasm. This approach, commonly referred to as "Precision Medicine," has led to the development of an ever-expanding repertoire of monoclonal antibodies and small molecular inhibitors. These agents effectively inhibit tumor cell proliferation and survival while avoiding the deleterious side effects on normal cell types seen with conventional chemotherapeutic agents and radiotherapy. Monoclonal antibodies used for the treatment of human cancers most commonly target cell surface molecules such as growth factor receptors¹ (e.g., the large family of membrane-spanning receptor tyrosine kinases) and immune response modulators² (e.g., programmed cell death protein 1, programmed death-ligand 1). Small molecular inhibitors can inhibit either cell surface proteins or signaling proteins that are located intracellularly³. However, to effectively employ these new therapeutic agents, it must be established that a particular cancer is dependent upon the molecule that is being targeted by a candidate therapeutic agent.

Although these new therapeutic agents have more focused effects, many of them still inhibit the action of more than one protein. In addition, multiple agents with varying effectiveness and specificity are often available to target a specific protein. Consequently, during preclinical investigations, it is wise to use additional approaches such as genetic ablation to validate a candidate protein as a therapeutic target. One especially useful approach to validating a protein as a therapeutic target is to ablate the gene encoding the candidate protein in a genetically engineered animal model that develops the specific cancer type of interest. This approach can be relatively straightforward if mice with a null mutation (either a natural mutation, a genetically engineered null mutation [a "knockout"], or a null mutation introduced by a gene trap) are available, and the mice are viable into adulthood. Unfortunately, mice with a null mutation that meet these criteria are often not available, typically because the null mutation results in death embryonically or in the first days of postnatal life. In this circumstance, mice prone to develop the tumor type of interest may instead be crossed to mice in which key segments of the gene of interest are flanked by loxP sites ("floxed"), which allows the gene to be ablated by introducing a transgene expressing Cre recombinase into the tumor cells (a conditional knockout). This approach provides several advantages. First, if a Cre driver is available that is expressed in the tumor but not in the cell type that led to death in conventional knockouts, this approach can potentially validate the candidate therapeutic target. Second, ablating the gene encoding the candidate protein in tumor cells but not in other intratumoral elements such as tumor-associated fibroblasts or immune cells allows the investigator to distinguish between cell-autonomous and non-cell-autonomous effects of the therapeutic target. Finally, a tamoxifen-inducible Cre driver (CreER^T2) allows the investigator to delete the gene of interest at different stages in tumor development and define the window in which the candidate therapeutic agent is most likely to be effective.

Unfortunately, there are also technical issues that can limit the use of conditional knockouts in tumors arising in GEM models. For instance, a Cre driver that is expressed in tumor cells and avoids gene deletion in normal cells essential for life may not be available. Another issue, which may be underestimated, is that Cre and CreER^T2 drivers often variably ablate floxed alleles in mice, resulting in mosaicism for the null mutation in a GEM cancer. When this occurs, tumor cells in which the targeted gene has not been ablated will continue to proliferate rapidly, overgrowing the tumor cells with ablated alleles. Mosaicism in Cre driver lines can occur due to non-ubiquitous Cre expression in the lineage targeted and by failed recombination in individual cells independent of Cre expression⁴. This is a known phenomenon of Cre drivers that is cell-type dependent and should be considered during experimental design and data interpretation. Mosaicism can mask the effect of the knockout and lead an investigator to erroneously conclude that the gene of interest is not essential for tumor cell proliferation and/or survival and thus is not a valid therapeutic target.

Several of these problems were encountered in a previous study that attempted to determine whether the receptor tyrosine kinase erbB4 was a potential therapeutic target in MPNST cells⁵. In these studies, mice were used that express a transgene encoding the neuregulin-1 (NRG1) isoform glial growth factor-β3 (GGFβ3) under the control of the Schwann cell-specific myelin protein zero promoter (P₀-GGFβ3 mice). P₀-GGFβ3 mice develop multiple plexiform neurofibromas that progress to become MPNSTs via a process that recapitulates the processes of neurofibroma pathogenesis and plexiform neurofibroma-MPNST progression seen in patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1)⁶. When crossed to mice with a Trp53 null mutation, the resulting P₀-GGFβ3;Trp53^+/- mice develop MPNSTs de novo as is seen in cis-Nf1^+/-;Trp53^+/- mice.

These MPNSTs recapitulate the progression from World Health Organization (WHO) grade II to WHO grade IV lesions seen in humans⁷. In P₀-GGFβ3 mice, MPNSTs arise within pre-existing plexiform neurofibromas in the trigeminal nerve (58%) and spinal dorsal nerve roots (68%)⁷; the MPNSTs arising in P₀-GGFβ3;Trp53^+/- mice have a highly similar distribution. In humans, MPNSTs most commonly arise in the sciatic nerve followed by the brachial plexus, spinal nerve roots, vagus, femoral, median, sacral plexus, popliteal obturator, and posterior tibial and ulnar nerves⁸. This tumor distribution in these GEM models is somewhat different from what is seen in humans. However, the MPNSTs that arise in P₀-GGFβ3 and P₀-GGFβ3;Trp53^+/- mice are histologically identical to human MPNSTs, carry many of the same mutations seen in human MPNSTs, and recapitulate the process of neurofibroma-MPNST progression seen in NF1 patients. The generation of P₀-GGFβ3 or P₀-GGFβ3;Trp53^+/- mice that were Erbb4^-/- was not feasible as mice with two Erbb4 null alleles die in utero at embryonic day 10.5 secondary to cardiac defects⁹. Because rescuing Erbb4 expression in the heart by introducing a cardiac-specific Erbb4 transgene (α-myosin heavy chain (MHC)-Erbb4) results in viable Erbb4^-/- mice¹⁰, the generation of mice with a complicated P₀-GGFβ3;Trp53^+/-;α-MHC-Erbb4;Erbb4^-/- genotype was attempted.

However, the matings did not produce mice in the expected Mendelian ratios, indicating that the desired genotype was deleterious. Therefore, the generation of P₀-GGFβ3;Trp53^+/- mice with floxed Erbb4 alleles¹¹ and a CreER^T2 driver was attempted to allow the deletion of Erbb4 in the MPNSTs arising in these mice. In these animals, numerous tumor cells with intact Erbb4 alleles were still present (mosaicism). The mosaicism observed could result from inefficient tamoxifen delivery, which resulted in differences in recombination efficiency within the tissue. The possibility of spontaneous compensatory mechanisms could further contribute to mosaicism in tamoxifen-mediated recombination by bypassing the requirement for Erbb4 expression. It is feasible that the loss of Trp53 makes tumor cells susceptible to additional spontaneous "permissive" mutations that could confuse the interpretation of the data. As it seemed likely that the Erbb4-intact MPNST cells would mask the consequences of ablating Erbb4 in other tumor cells, this approach was abandoned.

These obstacles led to the development of a methodology for ablating Erbb4 in very early passage MPNST cells using an adenovirus expressing Cre recombinase and eGFP. These cells can be separated from non-infected cells using FACS, which markedly reduces mosaicism for the ablated Erbb4 gene. Below, the methods used to achieve this, together with the methods used to assess the effects of gene ablation in vitro and in vivo, are described. The following protocol is an example of how to produce tumor-bearing mice that yield tumors carrying floxed alleles of embryonic lethal genes of interest for ex vivo excision prior to in vivo allograft tumor growth assessment. This includes a description of the approaches used to analyze the effect that Erbb4 ablation exerts on tumor cell proliferation, survival, and gene expression in vitro and proliferation, survival, and angiogenesis in orthotopic allografts.

Protocol

Prior to performing any procedures with mice, all procedures must be reviewed and approved by the Institutional Animal Care and Use Committee. The protocol described in this manuscript was approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. This protocol was performed by properly trained personnel following MUSC's institutional animal care guidelines.

1. Generation of mice that develop MPNSTs homozygous for Erbb4 ^flox alleles

1. Produce the F1 generation of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/+ animals by mating P₀-GGFβ3;Trp53^+/- mice⁷ with Erbb4^fl/fl mice¹¹. Use a Punnett square (Figure 1) to guide the breeding scheme to ensure that enough male and female F1 pups are generated with the desired P₀-GGFβ3;Trp53^+/-;Erbb4^fl/+ genotype.
2. Genotype F1 offspring by isolating genomic DNA from a tail snip collected in accordance with IACUC guidelines and then perform PCR using previously described primers to detect the P₀-GGFβ3 transgene¹², Trp53 wild type (+) and null (-) alleles⁷, and Erbb4^flox and Erbb4 wild-type alleles¹³.
   1. Isolate tail DNA using methods detailed on jacks-lab.mit.edu/protocols.
   2. Make the PCR reaction mix with 25 ng DNA, 0.25 nM dNTPs, 0.02 U/µL Taq, 0.5 µM of each primer, and 1x PCR buffer. Perform PCR reaction with a single 95 °C incubation (to melt the genomic DNA and activate Taq) for 1 min followed by 35 PCR cycles of 94 °C, 10 s (melting); 55 °C, 30 s (annealing); 72 °C, 40 s (extension) followed by a single 72 °C (extension) for 5 min. Store the reactions at 4 °C until ready to run on a 1.2-1.5% agarose gel.
      NOTE: Annealing temperature depends on the PCR buffer system used; performing an annealing temperature gradient to determine the proper annealing temperature is recommended.
3. Produce the F2 generation of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl animals by mating the appropriate F1 progeny (P₀-GGFβ3;Trp53^-/+;Erbb4^fl/+) with each other.
   NOTE: Figure 1 illustrates the Punnet square predictions used to calculate the expected number of F2 offspring with the desired genotype.
4. Identify animals with the desired P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl genotype as described in step 1.2.
5. Mate F2 progeny to each other to maintain the P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl colony and genotype all the pups. Confirm that the newly introduced floxed allele does not compromise survival or tumor latency. Achieve this by establishing cohorts (20 mice/cohort) of P₀-GGFβ3;Trp53^+/- and P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl mice and follow their survival and frequency of tumor occurrence.
6. Monitor animals several times per week until the experimental endpoint is reached (maximum allowable tumor size/humane endpoint approved by IACUC).
   1. During the weekly monitoring, assess body weight, normal social and grooming behavior, and tumor size measurements. Arrange for veterinary assessment in case of weight loss of >10% of body weight, social seclusion, and hunching.
   2. When a tumor-bearing mouse is identified and has reached its humane endpoint, humanely euthanize the animal using carbon dioxide inhalation followed by cervical dislocation and sterilely remove the tumor using a scalpel knife. Take tumor measurements by measuring length, width, and depth using a caliper and weight tumor (mass and volume). Work quickly to avoid autolysis.
7. Section the tumor into three sections using breadloaf style cuts with a scalpel knife under sterile conditions to generate tissue segments for formalin fixation/paraffin-embedding (FFPE), early passage culture generation, and flash-frozen material (Figure 2A).
   1. Ensure that Section 1 (for early passage culture generation, step 1.7.2) is approximately 10% of the total tumor volume, Section 2 (for fixation and IHC, step 1.7.3) is 70% of the total tumor volume, and Section 3 (for flash freeze, step 1.7.4) is 20% of the total tumor volume.
   2. Take section 1 of the tumor for the preparation of early passage cultures (see below).
   3. Fix section 2 of the tumor in 4% paraformaldehyde overnight at 4 °C and then embed in paraffin (formalin-fixed paraffin-embedded (FFPE) for diagnostic workup.
   4. Flash-freeze section 3 for analytical analyses (e.g., immunoblots to verify that protein encoded by the targeted floxed gene is appropriately expressed).
8. Stain 5 µm-thick FFPE tissue sections with hematoxylin and eosin (H&E) to confirm the tumor diagnosis by a qualified pathologist.If appropriate, perform immunohistochemical staining (IHC) to confirm the tumor diagnosis.
   1. Immunostain MPNSTs for S100 calcium-binding protein B (S100β), nestin, and sex-determining region Y (SRY)-Box transcription factor 10 (Sox10)-three markers that are expressed in both MPNSTs and Schwann cells (tumor cell origin)^5,6,14,15. Stain the tumors with antibodies recognizing erbB4, the protein encoded by the gene targeted for ablation in downstream steps.
   2. To confirm the diagnosis, have a qualified veterinary or human pathologist assess all stained tumor slides following WHO diagnostic and grading criteria^5,6,7,16.

2. Ex vivo ablation of floxed Erbb4 alleles in MPNST cells

1. Establish early passage cultures from freshly collected MPNST tissue by placing the tissue from section 1 (step 1.7.3) in 10 mL of ice-cold sterile phosphate-buffered saline (PBS) on ice and then transferring it to a sterile work area (Figure 2B)¹⁶.
   NOTE: All subsequent procedures are to be performed in a sterile tissue culture hood.
2. Mince the tumor tissue into 2-4 mm pieces and triturate 8-10 times in a 10 cm² treated tissue culture dish with 10 mL of growth medium. Culture these preparations in high glucose DMEM-10 growth medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 1% glutamine, 10 µg/mL streptomycin, and 10 IU/mL penicillin) supplemented with 10 nM neuregulin-1β (NRG1β) and 2 µM forskolin. Add forskolin at this stage to inhibit the growth of common contaminating cell types such as fibroblasts.
3. Maintain the mechanically dissociated tissue and cells for up to 3 days at 37 °C in a 5% CO₂ atmosphere to establish an early passage culture. Do not separate dispersed cells and remaining tissue fragments at this point.
4. Refresh the cultures with new medium every 3-4 days. Allow the tumor cells to proliferate until they are confluent.
5. Expand the early passage cultures by splitting the confluent cultures into several dishes. Remove the growth medium and wash cells with 1x dPBS. Then, add 1 mL of 0.25% trypsin for 2-5 min at room temperature per 10 cm² treated cell culture dish. Gently triturate the culture to facilitate detachment and generate a single-cell suspension.
   1. Terminate the trypsinization by adding 2 mL of DMEM-10 growth medium. Collect the trypsinized cell mixture and transfer it to a 5 mL sterile centrifuge tube. Pellet the cells by centrifugation for 5 min at 500 × g at room temperature.
   2. Resuspend the cell pellet in 5 mL of DMEM-10. Split the cells into two to four 10 cm cell culture dishes, each containing 10 mL of growth media (approximately 1 × 10⁶ cells per dish).
6. After 5 passages (repeating steps in 2.5), use growth medium without NRG1β and forskolin. Immunostain a sample of the culture with an anti-S100β antibody to verify that the culture is composed solely of tumor cells. Maintain the cells in DMEM-10 growth medium in all subsequent passages.
7. At the next passage, count the cells and freeze down a portion of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNST cells collected from confluent cultures (3 × 10⁶ cells/vial).
8. Plate P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl early passage MPNST cells at a density of 1.5 × 10⁶ cells per 10 cm treated cell culture dish in DMEM-10 growth medium.
9.  Day 0: (Approximately 12-16 h after plating), wash the adherent cultures with 2-4 mL of dPBS and infect with Ad5CMV-Cre/eGFP or Ad5CMV-eGFP at approximately 400 plaque-forming units (pfu)/cell in 10 mL of serum-free DMEM (i.e., 30 µL of 2 × 10¹⁰ pfu of virus per 10 cm dish). Ablate the floxed Erbb4 alleles using the adenovirus at the maximum tolerated virus concentration (multiplicity of infection, MOI), as determined empirically. Refer to Figure 2C for a schematic of the workflow for this ablation.
10. Day 1: Approximately 16 h later, rescue the cultures by adding 10 mL of DMEM-10 to the infection cocktail.
11. Day 2 - 3: FACS sort infected cells following the recommended protocol from the core facility to sort eGFP-positive cells approximately 48 h after infection. Prior to FACS sorting, briefly check cells for eGFP signal on a fluorescence microscope to ensure that the cultures have been efficiently infected (~50-100% positive cells). Return cells recovered after FACS to 10 cm tissue culture dishes; reserve one dish for genomic DNA isolation.
12. Day 4 - 5: After FACS sorting, let the cells recover in a tissue culture incubator for at least 24-48 h and then prepare the cells for in vitro cell-based analyses or in vivo grafting. Confirm Erbb4 deletion via PCR using genomic DNA isolated from a portion of the sorted eGFP-positive cells. Verify that eGFP-positive cells are ErbB4-negative by PCR or by immunostaining a sample of the culture with an anti-erbB4 antibody.
    1. Isolate genomic DNA from the cells using standard acid-guanidinium-phenol and chloroform-based DNA isolation method¹⁷.
    2. Perform standard PCR to distinguish floxed Erbb4 alleles and ablated Erbb4 alleles. Perform 40 cycles with 2 ng DNA, 20 µM primers 1 + 2 to produce a 250 bp Erbb4-null product and a 350 bp floxed product¹³. Perform both PCR and antibody-based approaches to confirm knockout when no reliable antibodies are available.
       ​NOTE: Cells are ready for in vitro cell-based assays as described below. Many types of downstream analyses can be performed with cells prepared in this manner. The purpose of the protocols presented below is to provide a few examples of in vitro and in vivo applications of these tumor cells post-ablation of the Erbb4 gene.

3. Proliferation and viability assays in MPNST cells with ablated Erbb4 alleles

1. Perform proliferation assays over the next seven days on sorted MPNST cells plated in a multiwell plate using an image-based automated cytometer.
   1. Day 6: Plate 2,000 cells per well in a final volume of 150 µL (~13,300 cells/mL) of growth medium in a 96-well plate. Perform measurements in triplicate for each experimental cohort and 4 daily endpoint reads (3 replicates x 2 conditions x 4 days).
   2. Days 7, 9, 11, 13: Stain cells with Hoechst and propidium iodide (PI) dyes and image them on an automated plate reader to count the number of live and dead cells in each well. To achieve this, simultaneously stain the cells with Hoechst dye to stain the nuclei of both living and dead cells and with propidium iodide (PI) to label the dead cells. Perform all reads at the same time every day or every other day.
      1. Add 50 µL of a 4x PI/Hoechst staining solution (4 µg/mL each) to each well quantified that day (e.g., Day 7 only), and incubate for 30 min at 37 °C in the tissue culture incubator. Keep the plate protected from light.
         NOTE: The staining concentration of Hoechst needs to be empirically determined and can range from 1 to 10 µg/mL depending on the cell type.
      2. Read the stained wells after incubation using the appropriate fluorescent channel on the automated cell imager. After reading, return the plate to the incubator for future reads on subsequent days (i.e., Days 9, 11, 13).
      3. Quantify the staining intensities based on the imaging system used and calculate the number of dead cells, living cells, and total cells using the following settings: blue channel (377/50 nm, Hoechst): total cells (live and dead); red channel (531/40 nm, PI): dead cells only; blue - red (total - dead) = live cells.
2. Perform the cell viability assay over three days, if desired, following the assay manufacturers' protocol.
   NOTE: Viability assays can be combined with proliferation assays by performing a triple stain including calcein AM (488/32 nm; green channel, specifically labels living cells), PI, and Hoechst. An apoptosis assay may also be performed using cultures set up as described above; the specific protocol will depend on the imaging system.
   1. Day 6: Plate 4,000 cells per well in a final volume of 150 µL in a 96-well plate in triplicate.
   2. Days 7 - 9: Add 2,000x bioluminescent cell viability assay reagents (Table of Materials), return the plate to the incubator, and read the plate 1 h later. Perform subsequent reads at the same time every day. For the bioluminescence (MTT) assays, perform the assay exactly as outlined in the manufacturer's instructions every 24 h for 72 h using a luminometer plate reader.

4. RNA-Seq analyses and identification of genes whose expression is altered by Erbb4 loss

1. Day 6: Isolate total RNA from sorted MPNST cells using standard acid-guanidinium-phenol and chloroform-based methods. For experiments designed to detect changes in mRNA levels between two cohorts, prepare total RNA from at least three biological replicates in each cohort.
   1. To eliminate events caused by the adenoviral vector or eGFP expression rather than Erbb4 loss, isolate RNA from three biological replicates of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNST cells transduced with Ad5CMV-Cre/eGFP and three biological replicates transduced with Ad5CMV-eGFP.
      NOTE: Isolated RNA must have an RNA integrity number (RIN) score ≥ 8 for RNA-Seq analysis. Ensure that the sequencing core determines the RIN of all samples prior to constructing libraries.
2. Use 100-200 ng of high-quality total RNA from each sample to prepare RNA-Seq libraries (work with the core sequencing facility for this step). Perform high-throughput sequencing using a next-generation DNA sequencer. For mRNA quantification, perform single-ended sequencing to generate Fastq files. Ensure a minimum of 50 million reads for each sample.
   NOTE: Refer to Figure 3 for a schematic illustrating the workflow used to process RNA-Seq data and to quantify the expression of differentially expressed transcripts. Sequence data, in the form of Fastq files, are subjected to quality control procedures; >80% of the reads should yield a Phred score of 30 to be considered appropriate for subsequent analysis. The sequences are also preprocessed (trimmed) using Trimmomatic¹⁸ to remove adaptor sequences and filter out low-quality reads.
3. Perform RNA-Seq alignment and analysis on the fastq generated files using any capable software program (e.g., DNAStar^19,20 or Partek²¹). Follow the program-specific steps using the default settings to align the fastq files to the mouse reference genome (GRCm38/mm10).
   NOTE: Using DNAStar as an example, the general workflow is described below (Supplemental Figure 1).
   1. Select the analysis method, RNA-Seq.
   2. Select the reference genome, Mouse.
   3. Upload the BED file if provided one by the sequencing core.
   4. Upload the fastq sequencing files, assigning them unique replicate names.
   5. Group-replicate the fastq files and designate them to a replicate set (i.e., GFP, CRE)
      NOTE: Each alignment program has its specific steps, and the user must consult with the program user guide for the program-specific protocol. The program will then generate gene-specific raw count data from these Fastq files.
4. Perform statistical and normalization analysis (DESeq2, EdgeR) on the raw count data using any capable software programs, as described in 4.3, with the Ad5CMV-eGFP sample set as the control data set and Ad5CMV-Cre set as the test data set to identify differential gene expression signals with robust statistical power^22,23.
   1. Select GFP fastq files as the control data set.
   2. Select DESeq2 as the statistical and normalization method .
   3. Start assembly and analysis.
      NOTE: Each alignment program has its specific steps, and the user must consult with the program user guide for the program-specific protocol. The program will then generate gene-specific raw count data from these Fastq files. The statistical and normalization analysis is a built-in step in many programs, as shown in the example here, within the sequence alignment protocol. If an alternative sequence alignment software program is used that does not offer this subsequent built-in step, perform the statistical and normalization analysis on the raw count data at the terminal level using the DESeq2 or EdgeR coding packages written in R, freely available for download on Bioconductor.org.
5. Use these approaches to identify changes representing at least a 1.5 fold increase or decrease relative to the control (in this case, cells transduced with the Ad5CMV-eGFP virus). Use only those differentially expressed genes (DEGs) determined to be statistically significant that show ≥1.5-fold changes and a p-value 0.05 or a padj of 0.1 for subsequent functional enrichment analysis.
   NOTE: This will generate a list of DEG genes and their statistical power for functional enrichment analysis.
6. Perform functional enrichment analysis on statistically significant Erbb4-mediated DEG identified in 4.4 with a ranked gene list file using any of the freely available web-based tools. Determine biological and pathway significance of Erbb4 gene loss through gene ontology datasets integrated in these open-access functional enrichment analysis tools^24,25,26,27 (see the Table of Materials for examples and Figure 3 for an example workflow using Panther).
   1. Export the data obtained in step 4.4 as a spreadsheet.
   2. Rank the data by log2 fold change, p/padj value, or both.
   3. Be sure to remove all non-statistically significant data.
   4. Export the ranked gene list with gene ID only as a .txt file and upload it to the website.
      NOTE: Use only statistically significant DEG (p/padj values < 0.05 or 0.1, respectively) to generate a ranked input gene list using log2 fold changes (highest to lowest), p/padj-values (lowest to highest) or both [(-log10(pval)*sign(log2FC)]. There are several approaches to generate a ranked gene list and is empirically determined for the dataset and the question being asked. The specific type of functional enrichment analysis tool being used can also assist in determining the appropriate type of rank gene list. For additional resources on RNA-Seq, see the following papers^28,29,30.

5. Orthotopic allografting of MPNST cells with ablated Erbb4 alleles and analysis of the effects of Erbb4 ablation

1. Before performing orthotopic allografting of sorted MPNST cells, ensure that all procedures are reviewed and approved by the Institutional Animal Care and Use Committee, and that all procedures are performed by properly trained personnel.
2. On the day of injection, remove low passage cells (approximately 85% confluency) from cell culture plates or flasks using a non-enzymatic cell dissociation solution (e.g., a mixture of chelators; see the Table of Materials) and count the cells using a hemocytometer. For orthotopic injections into the sciatic nerve, reconstitute the cells at 16,667 cells/mL (50,000 cells per 3 µL for each animal) in DMEM-10³¹. Keep the cells on ice.
   NOTE: Some cultures will not successfully establish grafts unless the cells are injected in low-growth factor basement membrane matrix. The requirement for a basement membrane matrix (10-50%) must be empirically determined.
3. Assess in vivo allograft growth potential in post-infected cells by injecting MPNST cells into the sciatic nerve of 8-10 anesthetized Hsd: Athymic Nude-Foxn1^nu mice per cohort (aged 4-8 weeks). For orthotopic injection of tumor cells, refer to Turk et al³¹. Here, a subcutaneous injection is demonstrated.
   NOTE: To determine the number of experimental animals needed for statistical significance, consult with a biostatistician or it is recommended to use G*Power3, a freely available software³².
4. Monitor the animals closely twice daily for the first week post-injection for any signs of pain. Thereafter, monitor the grafted mice three times per week for caliper measurements and assess body condition scores (BCSs) as previously described^5,31. As outlined in IACUC guidelines, euthanize animals with a BCS of 2 or less.
5. As grafted cells grow at different rates, determine graft times empirically using control (unmodified) cells before performing experiments described in this section. To collect the grafts at the experimental endpoint, euthanize the mice humanely using carbon dioxide inhalation followed by cervical dislocation as a secondary measure. Record the final volume and mass measurements of each graft.
   NOTE: As unmodified P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNST cells typically reach the maximum size allowed by IACUC within 30-45 days, a typical timeline is around 30-45 days after injection.
6. Isolate and section tissue as described in steps 1.6-1.8. Fix part of the graft tissue overnight in 4% paraformaldehyde (Figure 2) and then embed it in paraffin. Prepare 5 µm sections from the FFPE tissue and mount them on slides. Perform H&E staining and other necessary immunostaining to confirm that the graft tissue is composed of tumor cells as described in 1.8.1. Snap-freeze the rest of the tumor tissue for downstream analyses, such as validating the loss of Erbb4 expression and assessing the effects of Erbb4 loss on the expression of other Erbb family members.
   NOTE: Confirmation of tumor cells in the graft tissue must be done because immunodeficient mice are prone to develop neoplasms. In the case of small grafts, it is important to ensure that scar tissue or inflammation is not mistaken for a neoplasm.
   1. Perform standard IHC staining on the FFPE tissue to compare the expression of proteins of interest in allografts grown from Ad5CMV-eGFP and Ad5CMV-Cre/eGFP treated cells. Stain the excised graft tissue using erbB4-specific antibodies to confirm differences in in vivo erbB4 expression between the two experimental conditions. Determine the proliferative index via Ki67 staining and the level of apoptosis via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining.
      ​NOTE: Any protein target of interest can also be assessed via IHC. For example, assess vascular density by immunostaining allografts with an anti-CD31 antibody (1:50 dilution) to determine in vivo vascular microenvironment differences mediated by gene ablation. The number of vascular profiles per 40x field can be counted for quantification. For these IHC-based assays, follow standard IHC protocols for the staining procedures and the manufacturer's protocol for TUNEL staining. For quantification of images, use the ImageJ plug-in "automated counting of single-color image."

Representative Results

Figure 4 illustrates a typical result obtained when transducing P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl MPNST cells with either the Ad5CMV-eGFP adenovirus or Ad5CMV-Cre/eGFP adenovirus (Figure 4A). Cultures are viewed with fluorescence microscopy to identify eGFP-expressing cells and by phase-contrast microscopy to determine the total number of cells present in the same field at 10x (top) and 40x (bottom). The percentage of cells expressing eGFP can then be calculated if desired. Representative FACS output data are shown in Supplemental Figure S2. Following FACS, the percentage of eGFP-expressing cells will be markedly increased, with virtually all the cells in each field expressing eGFP. Typical PCR genotyping results expected after Cre-mediated gene ablation, depending on the transfection efficiency, are complete gene knockout (100% efficiency) or a heterogeneous population of induced and uninduced cells (<100% efficiency) compared to control transduction results (GFP only, fl/fl) (Figure 4B). Figure 4C illustrates the characteristic histopathology found in orthotopic allografts of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNSTs compared to the tumors derived from the original GEM animal models.

In these experiments, Erbb4 ablation resulted in decreases in cellular proliferation and cellular density in vitro and in vivo, increases in TUNEL labeling indices in vivo, and decreased vascular density in vivo. Figure 5 illustrates representative results obtained with an image-based automated cytometer showing decreased cellular proliferation in Ad5CMV-Cre ablated cells vs. control Ad5CMV-GFP transduced cells (Figure 5A); these experiments were performed with early passage cultured cells. Expression of ErbB4 via immunohistochemistry in P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNST cells transduced with Ad5CMV-Cre or eGFP virus was decreased in the resultant Ad5CMV-Cre transduced allografts compared to control Ad5CMV-eGFP treated allograft tumors (Figure 5B). Vascular density was assessed via immunoreactivity detection for CD31; vascular density can be seen to be markedly reduced in allografts of Ad5CMV-Cre transduced cells (Figure 5C). Signal intensity can be quantified, if desired, using ImageJ.

Figure 1: Punnet square calculations of the ratios of expected genotypes when generating and crossing P₀-GGFβ3;Trp53^+/- mice with Erbb4^fl/fl mice (F1). Punnet square calculations of the ratios of expected genotypes when generating and maintaining P₀-GGFβ3;Trp53^-/+;Erbb4^fl/+ with each other (F2). Abbreviation: fl = Floxed. Please click here to view a larger version of this figure.

Figure 2: Workflow of the processused to ablate floxed Erbb4 alleles in early passage cultures of MPNST cells derived from P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl mice. (A) Harvest the tumor expressing the floxed gene. (B) Create early passage cultures from the excised tumor. (C) Infect early passage cultures to ablate the floxed gene and perform downstream assays. Abbreviations: MPNST = malignant peripheral nerve sheath tumor; IHC = Immunohistochemistry; PFA = Paraformaldehyde; FFPE = Formalin-fixed paraffin-embedded; H&E = Hematoxylin and eosin; FACS = fluorescence-activated cell sorting; GFP = green fluorescent protein; K/O = knockout; PCR = polymerase chain reaction. Please click here to view a larger version of this figure.

Figure 3: Schematic illustrating the RNA-Seq workflow. The workflow is used to identify differentially expressed transcripts between control (GFP-induced) and Erbb4-null (Cre-induced) cells and the resultant biological significance. Abbreviations: GFP = green fluorescent protein; RNA-Seq = RNA sequencing. Please click here to view a larger version of this figure.

Figure 4: Representative transduction efficiency results assessed by microscopy in P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl MPNST cells. (A) Cells transduced with either Ad5CMV-eGFP adenovirus or Ad5CMV-Cre/eGFP adenovirus. (B) PCR results of potential gene populations post-Ad5CMV infection. (C) Characteristic histopathology comparing the tumors generated in the originating GEM models to the orthotopically allografted P₀-GGFβ3;Trp53^-/+;Erbb4^fl/fl MPNST cells via H&E staining. Images were taken at 40x. Isotype-matched negative control antibodies were used for control staining. Scale bars = 400 µm (A, upper panel); 100 µm (A, lower panel), 20 µm (C). Abbreviations: MPNST = malignant peripheral nerve sheath tumor; GFP = green fluorescent protein; BF = brightfield; FACS = fluorescence-activated cell sorting; Neg Ctrl = negative control; PCR = polymerase chain reaction; GEM = genetically engineered mouse; fl = floxed. Please click here to view a larger version of this figure.

Figure 5: Representative results obtained in cultured MPNST early passage cultures and orthotopically allografted MPNST cells following transduction with either Ad5CMV-eGFP or Ad5CMV-Cre/eGFP. (A) Proliferation assay of cultured MPNST cells demonstrating decreased cellular density in cells transduced with Ad5CMV-Cre/eGFP compared to those transduced with Ad5CMV-eGFP. Quantified data are graphed on days 1, 3, and 5 (left). Day 1 and Day 5 confluency images taken on the image-based automatic cytometer are shown on the right (Images taken at 4x magnification with brightfield microscopy). Error bars represent the standard error of the mean (SEM) of at least 3 different independent experiments. (B) Representative ErbB4 staining in allograft tumors transduced with Ad5CMV-eGFP or Ad5CMV-Cre/eGFP adenovirus taken at 20x magnification. Red channel (Alexa 564, CD31), blue channel (Hoechst, nuclei). (C) Representative images of vascular density in allografts of P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl MPNST cells transduced with Ad5CMV-eGFP versus Ad5CMV-Cre/eGFP virus. Images were taken at 40x. Isotype-matched negative control antibodies were used for control staining. Scale bars = 100 µm. Abbreviations: MPNST = malignant peripheral nerve sheath tumor; eGFP = enhanced green fluorescent protein; IgG = immunoglobulin G; NEG = negative control; CD = cluster of differentiation. Please click here to view a larger version of this figure.

Supplemental Figure S1: Schematic illustrating a sequence alignment and analysis workflow. The workflow used in DNAStar to align and analyze RNA-sequencing files (fastq files). Abbreviations: GFP = green fluorescent protein; RNA-Seq = RNA sequencing., DEG = differentially expressed genes. Please click here to download this File.

Supplemental Figure S2: Representative FACS data from early passage cultures using green fluorescent protein. (A) GFP-expressing cells are sorted by FACS after setting fluorescent gate parameters from non-infected cells. (B) After the appropriate detection gate is set, GFP-positive (transduced) cells are separated from GFP-negative (non-transduced) cells. Abbreviations: SSC-A = side scatter-area of peak; FSC-H = forward scatter-height of peak; FSC-W = forward scatter-width of peak; GFP = green fluorescent protein; FACS = Fluorescence-activated cell sorting. Please click here to download this File.

Discussion

The detailed methods presented here were developed using a GEM model of MPNSTs. However, if the tumor tissue of interest can be dispersed into individual cells, these methodologies are easily adaptable for various tumor types arising in GEMs. It is important to ensure that the floxed allele does not result in i) decreased survival that can make it difficult to obtain sufficient mice to screen for tumors, or ii) increased tumor latency that can make it difficult to obtain enough tumor-bearing mice. If the floxed allele does not affect survival, the survival of both cohorts will be equivalent. If it has no effect on tumor latency, equivalent numbers of tumor-bearing mice should arise in both cohorts with a similar time course.

Similar approaches have been used to study neurofibromas and MPNSTs using conditional knockouts and tamoxifen-inducible Cre animals to generate inducible conditional knockout animals^33,34. The difference between these studies and this one, aside from the use of tamoxifen for Cre induction as previously discussed, is that these studies focus on tumor development with a tissue-specific knockout of NF1. As this study and these other studies also use GEMM-generated tumors, the difference lies with the question being asked. In this protocol, we are not studying the role that NF1 loss plays in MPNST pathogenesis but are instead studying the role that a specific gene (Erbb4) in GEMM-generated MPNSTs exerts on in vivo tumor growth, which would otherwise be impossible due to being embryonic lethal. Furthermore, this protocol avoids the use of tamoxifen for those approaches requiring such parameters.

Others have studied established human MPNST cell lines in xenograft models³⁵. That study differs from this one in that the grafted cells used are well-studied but high-passage human MPNST cell lines, and it focuses on establishing drug responses in xenografts. Those researchers are not studying the role of a specific gene in these MPNST cells for in vivo growth and the effects of gene loss on drug response. Another study similar to this one was performed by Dodd et al., using adenovirus Cre recombinase (Ad-Cre)-mediated NF1 loss in a temporal and spatial dependent manner, again a powerful method for expanding MPNST translational efforts³⁶. The study focused on the role of NF1 and Ink4a/Arf loss, two very commonly co-deleted genes in human MPNSTs, in the sciatic nerve by Ad-Cre injection. The P₀-GGFβ3;Trp53^+/-;Erbb4^fl/fl mice described in this study could be used in this fashion by injecting Ad-Cre into Schwann cell precursor lineages. However, the questions asked in this study are not focused on the evolutionary role of Erbb4 on MPNST initiation but on how MPNSTs may hijack Erbb4 signaling for tumor growth advantage.

There are alternatives to the approach described here, including knocking down gene expression via RNA interference (RNAi) or inactivating the gene of interest with CRISPR in human cancer cell lines. These alternative approaches are valuable and can be partnered with gene ablation in cells derived from GEM tumors to verify that the effects observed in mouse tumor cells are recapitulated in their human counterparts. However, there are some clear advantages to the methodology described here. First, genetically ablating floxed genes in GEM cancer cells is rapid and produces complete inactivation of the targeted gene. In contrast, it is often observed that the use of short hairpin RNAs results in only a partial knockdown of gene expression. Although CRISPR can produce complete gene inactivation, the identification of clones that have complete inactivation requires a prolonged selection process.

Further, multiple clones must be examined to ensure that the effects resulting from CRISPR ablation do not differ between different clones. Second, the ability to ablate floxed alleles in early passage GEM tumor cultures is advantageous as this minimizes the possibility of selecting for distinct tumor cell subpopulations that grow effectively in culture. In comparison, human cell lines, which have typically been maintained in culture for years, are often derived from distinct subpopulations that are well adapted for culture and frequently undergo genetic drift during this process. This results in these cell lines developing new mutations that were not present in their parent tumor. Finally, although the grafting of GEM tumor cells into immunodeficient mice is described in this manuscript, it is noted that GEM tumor cells derived from tumors arising in inbred mouse strains can be allografted into mice of the same strain. This would allow the investigator to assess tumor biology in animals with an intact immune system in the presence or absence of the targeted gene.

The critical step in the protocol described here is the ablation of the floxed gene with Ad5CMV-Cre/eGFP and ensuring the survival of the tumor cells following gene ablation and FACS. As this is a process that is dependent on the ability of Cre recombinase to ablate the floxed gene, it is potentially subject to some of the same problems that can be encountered when ablating a floxed gene in vivo; these issues would manifest as an incomplete ablation of the targeted allele. Note that the ability to purify cells that have been successfully transduced with the eGFP-expressing adenoviral vector does appear to minimize mosaicism compared to in vivo gene ablation with CreER^T2. However, in the case of targeted genes encoding proteins, such as ErbB4 that are expressed on the cell surface, it may be possible to further ensure the loss of the targeted gene by performing FACS utilizing both eGFP and an antibody recognizing the protein of interest (i.e., sort the eGFP-positive/erbB4-negative cells).

Because, as was noted in the introduction, many of the proteins targeted therapeutically by monoclonal antibodies are cell surface proteins, this approach should frequently be feasible. It should also be noted that it is important to let the tumor cells recover for at least 2-3 days after FACS if RNA-Seq is to be performed on them to assess the effect that gene ablation has had on the transcriptome. RNA-Seq datasets obtained very soon after FACS often show more variability in gene expression between biological replicates in some cell types. This may be a consequence of the trauma that cells suffer when going through FACS; hence, the cells must be allowed a recovery period after this process. RNA-Seq datasets can be analyzed using DESeq2, which uses the negative binomial distribution and a shrinkage estimator for the distribution's variance. In these analyses, samples are sorted according to their q-value, which is the smallest false discovery rate (FDR) at which changes in transcript levels are significant; FDRs are calculated using the Benjamini-Hochberg multiple testing adjustment procedure. Alternatively, DEGs can be identified using other available software capable of RNA-Seq analysis workflows.

For this MPNST animal model, identifying the experimental endpoint requires an understanding of the natural history of the neoplasms in the animal model that is being used. The natural history of MPNSTs in this GEM was determined by following the survival of a series of 44 P₀-GGFβ3 mice⁶ and 19 P₀-GGFβ3;Trp53^+/- mice⁷ and then performing complete necropsies on all of these animals when they reached their survival endpoint. The typical survival times and the location where MPNSTs typically arose were determined in each animal model. In most of these mice, MPNSTs arose in the trigeminal nerve. MPNSTs arising in the trigeminal nerve typically produced ptosis associated with corneal cloudiness (a result of problems with tear distribution produced by the ptosis), which provided a clinically useful sign to identify tumor-bearing mice. Tumor diagnosis should be confirmed by performing H&E stains and several additional diagnostic stains (S100β, nestin Sox 10, H&E, Mart1) followed by an examination by a qualified veterinary or human pathologist. This is also important because the Trp53 null allele in this model predisposes the mice to the development of other neoplasms such as lymphomas, and these tumors must be distinguished from MPNSTs.

Finally, this methodology has several potential applications not yet explored but may be feasible in future investigations. For example, neuregulin 1 (NRG1), the ligand that activates both erbB3 and erbB4, promotes the migration of MPNST cells in a chemotactic fashion; both erbB3 and erbB4 are located in the invadopodia of MPNST cells³⁷. However, it is not clear whether erbB3 or erbB4 mediates the chemotactic response to NRG1. Consequently, cells in which erbB4 is ablated with this methodology can be used to address this question. A natural follow-up to these experiments is to assess whether erbB4 is required for metastasis using the commonly utilized approach in which tumor cells are injected via the tail vein and the number of pulmonary metastases then assessed. This methodology offers a clear advantage over other approaches, such as using shRNAs to knock down erbB4 expression because shRNA expression is often silenced in cells after a while. Further, early passage cultures are anticipated to contain a mixture of tumor-derived subpopulations, including subpopulations of cells prone to metastasis. Performing metastasis assays with cells prepared using the methodology described here will allow an assessment of metastatic potential that is more representative of the parent tumor than an established cell line. This is, of course, just one example of the future applications that could utilize this methodology. We hope that other investigators will combine this methodology with problems of particular interest to enhance the ability to identify proteins that are key therapeutic targets in human cancers.

Materials

Name	Company	Catalog Number	Comments
Ad5CMV-eGFP	Gene Transfer Vector Core, Univ of Iowa	VVC-U of Iowa-4
Ad5CMVCre-eGFP	Gene Transfer Vector Core, Univ of Iowa	VVC-U of Iowa-1174
alexa 568 secondary antibody	Thermo/Fisher	GaR A11036
Bioconductor Open Source Software for Bioninformatics	Bioconductor	http://www.bioconductor.org	alternative statistical analysis tool used for step 4.4
CD31	Abcam	ab28364
Celigo Image Cytometer	Nexcelom Bioscience	N/A
Cell Stripper	Corning	25-056-Cl	mixture of chelators
DAB staining kit	Vector Labs	MP-7800
DAVID (Database for Annotation, Visualization, and Integrated Discovery)	DAVID	https://david.ncifcrf.gov	functional enrichment analysis software used for step 4.5
DMEM	Corning	15-013-Cl
DreamTaq and Buffer (Genotyping PCR)	Thermo/Fisher	EP0701 and K1072
erbB4 antibodies	Santa Cruz	sc-284
erbB4 antibodies	Abcam	ab35374
erbB4 antibodies	Millipore	HFR1: 05-1133
FACS Sorter	BD Biosciences	Aria II
Forskolin	Sigma	F6886
GenomeSpace Tools and Data Sources	GenomeSpace	https://genomespace.org/support/tools/	general resource for several types of open source bioinformatic tools for step 4.5
Glutamine	Corning	25-005-Cl
Gorilla Gene Ontology enRIchment anaLysis and visuaLizAtion tool	Gorilla	N/A, http://cbl-gorilla.cs.technion.ac.il	functional enrichment analysis software used for step 4.5
GSEA Gene Set Enrichment Analysis	Broad Institute	N/A, https://www.gsea-msigdb.org/gsea/index.jsp	functional enrichment analysis software used for step 4.5
HSD: Athymic Nude-FOxn1nu mice	Envigo (Previously Harlan Labs)	69
Illumina HiSeq2500 (next generation DNA sequencer)	Illumina	Hi Seq 2500	DNA sequencer used for step 4.2
Lasergene: ArrayStar Gene expression and variant analysis	DNAStar LaserGene software	N/A	software statistical and normalization analysis used for step 4.4
Lasergene: SeqMan NGen sequence alignment assembly	software alignment used for step 4.3	N/A	software alignment used for step 4.3
Matrigel, low growth factor basement membrane matrix	Corning	354230
NRG1-beta	In house	Generated by SLC, also commercially available from R & D Systems(396-HB-050/CF).
Nuclear Stain Hoeschst 33342	Thermo	62249
Panther Gene Ontology Classification System	Panther	http://pantherdb.org	functional enrichment analysis software used for step 4.5
Partek (BWA aligner and analyzer)	Partek, Ver 7	N/A	software alignment and statistical/normalization used for step 4.3
Pen/Strep	Corning	30-002-Cl
Primer 1: 5′-CAAATGCTCTCTCTGTTCTTTGT GTCTG- 3′	Eurofins Genomics	Primer 1 + 2: 250 bp ErbB4 null product and a 350 bp Floxed ErbB4 product;
Primer 2: 5′-TTTTGCCAAGTTCTAATTCCATC AGAAGC-3′	Eurofins Genomics	Primer 1 + 2: 250 bp ErbB4 null product and a 350 bp Floxed ErbB4 product;
Primer 3: 5′-TATTGTGTTCATCTATCATTGCA ACCCAG-3′	Eurofins Genomics	Primer 1 + 3: 350 bp wild-type ErbB4 product.
Propidium Iodine	Fisher	51-351-0
Proteom Profiler Phospho-Kinase Arrays	R&D Systems	ARY003B
Real time glo	Promega	G9712	bioluminescent cell viability assay
ToppGene Suite	ToppGene	https://toppgene.cchmc.org	functional enrichment analysis software used for step 4.5
Trizol (acid-quanidinium-phenol and choloroform based reagent)	Invitrogen	15596026
Tyramide Signal Amplification Kit	Perkin Elmer	NEL721001EA